Figure 2

MMS22Linactivation impairs in vitro erythropoiesis. (A) Cell proliferation of shMMS22L and shCTRL‐transduced erythroid progenitors was monitored for 96 h by real‐time videomicroscopy using the Incucyte® system. Results are represented as the fold increase of cell confluence compared to Day 0 and show mean ± SD for three technical replicates. Data are representative of three independent experiments. (B) Cell cycle analysis of shMMS22L and shCTRL‐transduced erythroid progenitors. Percentage of cells in each phase of the cell cycle is determined by FACS analysis at Day 6 of differentiation. Results are represented as the mean ± SD of three independent experiments. (C) Percentage of apoptotic shMMS22L and shCTRL‐transduced cells determined by FACS analysis as the percentage of Annexin V positive cells amongst the PI negative population at Day 4 and Day 6 of differentiation. Results are represented as the mean ± SD of three independent experiments. (D) Erythroid differentiation of shMMS22L and shCTRL‐transduced erythroid progenitors was assessed by FACS as the percentage of GPA‐positive cells at Day 5, Day 7, Day 9, and Day 11 of differentiation. Results are represented as the mean percentage ± SD of three independent experiments. (E) Cell proliferation of shMMS22L and shCTRL‐transduced CD36‐positive and CD36‐negative cells was monitored for 120 h by real‐time videomicroscopy using the Incucyte® system. Results are represented as the fold increase of cell confluence compared to Day 0 and show mean ± SD for three technical replicates. (F) Percentage of apoptotic shMMS22L and shCTRL‐transduced cells differentiated toward the erythroid or the graulo‐monocytic lineage, determined by FACS analysis as the percentage of Annexin V positive cells amongst the PI negative population at day 6 of differentiation. Results are represented as the mean ± SD of three independent experiments. (G) Erythroid differentiation of shMMS22L and shCTRL‐transduced CD36 positive cells was assessed by FACS as the percentage of GPA‐ positive cells at day 12 of differentiation. Granulo‐monocytic differentiation of shMMS22L and shCTRL‐transduced CD36‐negative cells was assessed by FACS as the percentage of CD11B and CD16 positive cells at Day 12 of differentiation. (H) Representative images of MGG‐stained cells at Day 7 of erythroid (top panel) or granulo‐monocytic (bottom panel) differentiation. Basophilic and polychromatic erythroblasts can be observed in the shCTRL transduced CD36+ cells, whereas shMMS22L‐tranduced cells, CD36+ resemble granulocytic and monocytic progenitors. In both shCTRL and shMMS22L‐transduced CD36− cells, progenitors differentiate into mature macrophages and granulocytes. P‐values are determined by a two‐tailed t‐test. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.